There is much current interest in the role of CYP2E1 in alcohol-induced liver injury. We propose to characterize CYP2E1- and ethanol-mediated cytotoxicity in HepG2 cell models either transduced or transfected to express human CYP2E1. We have recently developed a new HepG2 cell line which over-expresses CYP2E1 at levels 5 to 10-fold greater than previously established cell lines. The CYP2E1 over-expressing E47 cells grow at a slower rate than control cells but remain viable. When GSH is depleted, marked toxicity is observed with the E47 cells but not control cells. Sp. Aim I is designed to characterize and evaluate mechanisms involved in this growth inhibition effect caused by over-expression of CYP2E1 and in the cytotoxic effect observed when GSH is depleted. The ability of antioxidants and iron chelators to prevent the growth inhibition and cytotoxicity, and the ability of iron and polyunsaturated fatty acids to exacerbate these effects will be determined. Impairment of mitochondrial function and development of oxidative stress will be studied. Whether injury is apoptotic in nature and the ability of bcl-2 to "rescue" the cells from CYP2E1-catalyzed toxicity will be evaluated. Calcium plays a critical role in cell injury produced by oxidative stress and hepatotoxins. Aim II will evaluate the ability of metabolites derived from CYP2E1 oxidation of ethanol and other substrates and CYP2E1-catalyzed formation of reactive oxygen species to activate Ca2+ channels in microsomes and promote release of Ca2+. The role of Ca2+ in toxicity exhibited by ethanol, PUFA or over-expressed CYP2E1 in HepG2 cells will be evaluated. Aim III will evaluate whether oxidative stress mediated by CYP2E1 itself or CYP2E1 interactions with ethanol, PUFA, acetaminophen activates the transcription factor NF-kB, and if so, whether the HepG2 cells can initially be protected against injury by upregulation of NF-kB-activated genes. Special emphasis will be on cellular GSH levels. TNF-CYP2E1 interactions and induction of IL-8, a powerful chemoattractant will be evaluated. Selected experiments will be carried out with cultures containing hepatocytes isolated from chronic ethanol-fed rats and pair-fed controls to allow extrapolation of results with the HepG2 cells to normal hepatocytes. It is hoped that these studies will help to define the role of CYP2E1 and CYP2E1-derived reactive oxygen species in the hepatotoxic actions of ethanol.